CN1278267A - Human thrombopoietin produced established human ccell lines, process for producing same, and medicinal compositions contg. same - Google Patents

Abstract

Human thrombopoietin (TPO) similar to natural sugar chain structues produced by established human cell lines originating in human TPO-producing organs (for example, human liver-derived cells and human bone marrow stroma cells); a process for producing TPO which involves the step of expressing a foreign human TPO gene in an established human cell line; medicinal compositions containing TPO, such as remedies for thrombopenia; and human TPO having at least a sugar chain structure of the alpha -2,6-bond type sialic acid. This TPO can contribute to the improvement in depression of antigenicity, internal dynamics, etc.

Description

Human thrombopoietin, its preparation method that human cell line produces and comprise the pharmaceutical composition of this material

Background of invention

Invention field

The present invention relates to the Novel Human thrombopoietin glycoprotein that human cell line produces, particularly relate to and to reduce antigenicity and improve Novel Human thrombopoietin albumen, its preparation method of body internal dynamics and comprise the pharmaceutical composition of this material.

Human thrombopoietin (thrombopoietin is called for short TPO) is the glycoprotein that clones as the part of Mpl, and Mpl is one of cytokine receptor kinsfolk (de Sauvage etc., Nature (London), 369 volumes, 533-565 page or leaf, (1994); Bartley, T.D. etc., Cell, 77 volumes, 1117-1124 page or leaf, (1994)).These Mpl parts all can (for example, people, mouse, dog etc. detect in) serum and the blood plasma, determine that therefore they are relevant with hematoblastic formation with megalokaryocyte the thrombocytopenia animal.

Therapeutical agent with the exploitation thrombocytopenia is a target, the inventor is to promote that generating Megakaryocytic activity by the highly purified macronucleus precursor cell of rat marrow is index, from the rat plasma of thrombocytopenia purifying the TPO of rat, on the basis of its partial amino-acid series, cloned the TPO cDNA of rat, further cloned people's TPO cDNA, people TPO (the H.Miyazaki etc. of a large amount of homogeneous have successfully been obtained by gene recombination technology, experimental hematology, 22 volumes, 838 pages, (1994)).Verified, the people TPO that the inventor successfully obtains and the factor that the front is obtained as people Mpl part have identical aminoacid sequence (with reference to the sequence table of back: sequence number 1).

The inventor gives this people TPO to find during with the mouse that presses down cancer agent and immunosuppressor or cause myelosuppressive thrombocytopenia by (radiation exposure) and BMT because of convenient, it can stop thrombopenia, promote thrombocyte to increase, and seeing the hyperfunction of hemopoietic function, this people TPO is effective to these thrombocytopenias.

At present about people TPO, the report of TPO in the existing human plasma (Matsumoto, A is etc., the 38th ASH meeting (1996)), but owing to separated, the material of purifying, so its structure function the unknown.

In addition, can come out at mRNA and ELISA level detection by the TPO that various cell produced.With the liver cell that derives from the people (HepG2) (Hino, M. etc., biological chemistry and biophysical research communication, 217 volumes, 475-481 page or leaf, (1995)), can detect composing type mRNA.In addition, personnel selection fetal kidney cell (HEK) can detect TPO mRNA, and it has the mpl of making express cell (mo7e, BaF3/mpl) Zeng Zhi activity.But still fail to its separate, purifying, comprise activity in vivo, its structure, function are not still known.

On the other hand, the recombinant human TPO that the applying gene recombinant technology is produced can express (Kato in cercopithecus aethiops kidney cell (COS-1), T. etc., biochemical magazine, 118 volumes, the 229-236 page or leaf, (1995)), in Chinese hamster ovary cell (CHO), express (Morita, H. etc., FEBS Lett 395 volumes, 228-334 page or leaf, (1995), Kato.T. etc., institute of NAS periodical, 94 volumes, 4669-4697 page or leaf, (1997)), at intestinal bacteria (Ann.M.F. etc., blood, 86 volumes, the 54-59 page or leaf, (1995)), little hamster kidney cell (BHK) (Ross, C.H. etc., biological chemistry, 35 volumes, 14849-14861 page or leaf (1996)), human fetal kidney cell (HEK 293) (de Sauvage etc., nature, 369 volumes, 533-538 page or leaf (1994); Bartley, T.D. etc., cell, 1117-1124 page or leaf, (1994)) the middle expression.

But, up to the present also not about express the report of TPO with the human cell line that derives from TPO generation organ.

In addition, by the TPO that above-mentioned various non-human cell line produced, what similarities and differences the people TPO and the TPO in the human blood that produce with human cell line structurally have do not understand as yet.Therefore, how the relation between the difference of its structure and TPO function does not appear in the newspapers as yet at present.

Generally speaking, glycoprotein with TPO sample physiologically active except that main TPO produces internal organs, also has other to produce these proteinic internal organs, owing to derive from the kind difference of this, the sort of internal organs or cells of organs, sometimes the structure difference of this sugar-protein that is produced.

Equally, with the recombinant glycoprotein of gene recombination technology production, since used host cell difference, the sugar chain structure difference that it produced.When especially making host cell with zooblast, the sugar chain structure that (animal species or come source organ's kind, cell strain etc.) added because the difference of its kind is varied, this species diversity is given proteinic function, mainly is that antigenicity, body internal dynamics etc. are brought many influences.In fact, the report (Steis etc., N.Eng.J.Med.318 volume, 1409-1413 page or leaf (1988) etc.) that a lot of gene recombinant protein medicines is produced antibody is arranged at present, this for no other reason than that they constructing, there are differences on the function and to cause with intravital native protein.

If have the more people TPO glycoprotein of advantageous property aspect having in antigenicity, body dynamically, estimate the higher farmland of its value as medicine.

Brief summary of the invention

The invention provides the human thrombopoietin (TPO) that human cell line produces.

In embodiment of the present invention, TPO is the expression product in human cell line by endogenous or ectogenic people TPO gene, separated, the purifying of product.In addition, recommend TPO to produce the cell strain in internal organs source as human cell line, for example JHH7 (FERM BP-6049), HuH7 (FERM BP-6048) etc. derive from the cell and the human bone marrow matrix oncocyte of people's liver.

TPO of the present invention contains the sugar chain structure that has affinity with SSA lectin post, perhaps, contains the sialylated sugar chain structure of α 2,6 connecting-types at least.Its aminoacid sequence has the amino acid of 1-332 position as shown in the sequence number 1 of sequence table.

It is the pharmaceutical composition of effective constituent that the present invention also provides the people TPO with above-mentioned definition.Particularly, this TPO can be used as the thrombocyte dose, the agent of thrombocyte treating dysfunction, thrombopenia Remedies.When TPO of the present invention uses as medicament, can have and reduce antigenicity and improve in the body advantage such as pharmacokinetics.

The present invention also provides the preparation method of people TPO, and it is included in and derives from the ectogenic people TPO gene of the interior expression of human cell line that TPO produces internal organs.Can use the cell (for example JHH7 (FERM BP-6049) etc.) and the human bone marrow matrix oncocyte of the above-mentioned people's of deriving from liver as human cell line.Also be contained among the present invention with the people TPO that this method obtained.

The present invention also provides the people TPO that has the sialylated sugar chain structure of α 2,6 connecting-types at least.The people TPO here can be expression product, product separated, the purifying of ectogenic people TPO gene in cell strain.And can be the Chinese hamster ovary celI that has imported α 2,6 sialic acid based transferase genes as cell strain.

In addition, the present invention also provides has α 2 at least, the preparation method of the people TPO of 6 connecting-type saliva acidic group sugar chain structures, it comprises α 2,6 sialic acid based transferase cDNA import in the Chinese hamster ovary celI, then expressing human TPO gene in this Chinese hamster ovary celI, perhaps expressing human TPO gene in Chinese hamster ovary celI, then α 2,6 sialic acid based transferase cDNA are imported the preparation method of this Chinese hamster ovary celI.

The accompanying drawing summary

Fig. 1 is by relatively the recombinate photo of ChoTPO, natural HepTPO (JHH7) and natural HepTPO (HuH7) molecular weight of electrophoresis.Detection is behind electrophoresis, carries out the reaction with anti-TPO sheep IgGXRAG-HRP.Each swimming lane TPO of 250pg.

Fig. 2 is by relatively the recombinate photo of molecular weight of HepTPO (JHH7/7/c83), natural HepTPO (JHH7) and recombinant C hoTPO of electrophoresis.Detect with Fig. 1 and carry out equally.

Fig. 3 uses each TPO shown in the lectin ELISA method comparison diagram and the reactivity of SSA lectin or MAM lectin.

Fig. 4 is an external activity of using the TPO shown in the FDCP-hMp1635 analytical method demonstration figure.

Fig. 5 relatively clones CHO+2 by electrophoresis/Western trace (using anti-hTPO sheep polyclonal antibody and the anti-sheep IgG of horseradish peroxidase bonded rabbit polyclonal antibody), 6ST/19/c4 and CHO+2, the TPO that 6ST/21/c3 produces is with the photo of the recombinant C hoTPO molecular weight of cloning the CHO29c14 generation.

Fig. 6 represents with lectin-ELISA method analysis clone CHO+2,6ST/19/c4 and CHO+2, the sialic result of the TPO that 6ST/21/c3 produces.

Fig. 7 represents to use TPO that Chinese hamster ovary celI (representing with 19c4 and 21c3 among the figure) that the FDCP-hMp1635 assay method relatively imported α 2,6 sialytransferase cDNA and CHO D-cell (representing with HTCF among the figure) express in external activity.

Detailed Description Of The Invention

Owing to verified that in the mRNA level the main generation internal organs of hTPO are liver (Shimada, Y. etc., experimental hematology, 23 volumes, 1388-1396 page or leaf, (1995); Nomura, S. etc., experimental hematology, 25 volumes, 565-572 page or leaf, (1995)), the cell line of people's liver so the inventor will concentrate on to originate. So preparation is from the TPO (native plasma TPO) of human plasma fraction's purifying, derive from the TPO (natural HepTPO) of liver cell strain purifying and the restructuring hTPO (restructuring HepTPO) of being expressed by the cell line that produces this hTPO and derive from liver from producing hTPO, with the restructuring hTPO (recombinant C hoTPO) of expressing among the CHO their sugar chain structure relatively. The result shows, produce TPO (natural HepTPO) that the cell line derive from liver is purified into and this hTPO from hTPO and produce the restructuring hTPO (restructuring HepTPO) that the cell line that derives from liver is expressed, the combination that its N connects sugar chain is different from the restructuring hTPO of expressing cho cell, but identical with the TPO that is purified into from human plasma (native plasma TPO).

That is to say, as described later shown in the embodiment, use anti-TPO monoclonal antibody and agglutinin and carry out to find when agglutinin ELISA method is analyzed that native plasma TPO, natural HepTPO and restructuring HepTPO are the SSA agglutinin (Shibuya that α 2,6 connects with the sialic connected mode of judgement sugar-chain end, N. etc., journal of biological chemistry, 106 volumes, 1098-1103 page or leaf, (1989)) have reactivity, but recombinant C hoTPO do not have reactivity; On the other hand, for judging that the sialic acid connected mode is the MAM agglutinin (Wei-Chun, W. etc., journal of biological chemistry, 263 volumes, 4576-4585 page or leaf, (1988)) that α 2,3 connects, they all have reactivity. In a word, the TPO that derives from human plasma that so to say that has α 2,6 connection sialic acid structures in recombinant C hoTPO and the N connecting-type sugar chain has very big-difference, and natural HepTPO and restructuring HepTPO are similar with the TPO that derives from human plasma.

So, with reference to the present invention, can provide the Novel TPO painting that is produced by human cell line. Preferably provide by TPO and produce the novel hTPO that the human cell line in internal organs source produces. Novel TPO painting with above-mentioned specific sugar chain structure more preferably is provided.

Novel hTPO of the present invention can reduce antigenicity, improves the pharmacokinetics in the body, improves the reduction of the thrombocythemia effect that causes because of continuous administration.

The product that the hTPO gene of hTPO recommendation of the present invention exogenous (exogenous) is expressed in human cell line, and ectogenic hTPO gene is the applying gene recombinant technique, with the strain of hTPO gene transfered cell, make it to express, separation, purifying obtain. Perhaps use the expression product of TPO gene in human cell line of endogenous (endogenous) as hTPO, and this endogenic TPO gene is that separation, purifying obtain from the human cell line of expressing the hTPO gene. Expression product as endogenic hTPO gene, also comprise the hTPO that obtains with reference to next method, be about to importings such as promoter and contain in the human cell line of TPO gene, make this endogenic TPO gene activation, expression (world discloses WO96/29411 number).

As human cell line, can recommend such as HL60 (ATCC preserving number CCL-240), Jurkat (ATCC preserving number TIB-152), K562 (ATCC preserving number TIB-152), HeLa (reason is ground preserving number RCB0027), HepG2 (ATCC preserving number CRL10741) etc. Advantageous applications derives from the human cell line that TPO produces internal organs, such as the cell that derives from people's liver, human bone marrow matrix oncocyte etc. More preferably derive from cell line JHH7, HuH7, HepG2 of people's liver etc. JHH7 is preserved in Japanese trading industry on August 11st, 1997 and economizes Industrial Technology Institute life engineering Industrial Technology Research Institute according to budapest treaty, and preserving number is FERM BP-6049. Same HuH7 is preserved in Japanese trading industry on August 11st, 1997 and economizes Industrial Technology Institute life engineering Industrial Technology Research Institute, and preserving number is FERM BP-6048.

Preferred hTPO of the present invention, have 2, the 6 sialylated sugar chain structures that connect, perhaps comprise at least and (the regular title reference: blue sieve Piao's (Sambucus sieboldina) aggulutinin agglutinin or EIderberry Bark agglutinin (Sibuya of SSA agglutinin, N. etc., journal of biological chemistry, 262:1596-1616 (1987)) sugar chain structure that has compatibility. More preferably in N connecting-type sugar chain, contain the sialylated sugar chain structure that α 2,6 connects, or the sugar chain structure that has compatibility with the SSA agglutinin.

People TPO of the present invention, the primary structure of its protein portion, be that its aminoacid sequence comprises the aminoacid sequence shown in the sequence number 1, perhaps keep people TPO activity and in the aminoacid sequence part change (displacement, disappearance, insert and/or add) sequence.Particularly, can recommend to resemble international TPO derivative of WO95/21919 number (August 17 nineteen ninety-five) record and the TPO derivative of WO96/25498 number (on August 22nd, 1996) record of disclosing, WO95/18858 number (July 13 nineteen ninety-five), the TPO derivative of record carried out the change of amino-acid residue like that.

In this specification sheets, " TPO activity " is meant the propagation and the differentiation that can promote the macronucleus precursor cell, and/or differential stimulus or strengthen the activity that thrombocyte produces in vivo.

In addition, the present invention also provides the preparation method of above-mentioned Novel Human TPO.

As from human cell line direct purification and isolating method, can use applied conventional means in the protein purification, for example ion exchange chromatography, lectin affinity chromatography, pigment adsorption chromatography, hydrophobic interaction chromatography, gel permeation chromatography, reversed phase chromatography, heparin affinity chromatography, sulfation gel chromatography, hydroxyapatite chromatography, metallo-chelate chromatography, iso-electric point chromatography, separation electrophoresis and iso-electric point electrophoresis etc., but several different methods applied in any combination.In addition, the method that the TPO physico-chemical property can make up of should choosing is also included among the present invention.Also can use the antibody column of the Antibody Preparation that can discern TPO.In addition, be part (de Sauvage etc., Nature, 369 volumes, 533-565 page or leaf, (1994): Bartley, T.D. etc., Cell, 77 volumes, 1117-1124 page or leaf, (1994) of Mpl because proved TPO; Kaushanaky etc., Nature, 369 volumes, 565-568 page or leaf, (1994)), make Mpl activatory glue can prepare the affinity gel column by coupling, but utilize this point purifying TPO.More specifically, can recommend to do the host, by the Mpl-X post (aforesaid Bartley etc. (1994)) for preparing of cell extracellular region (Mpl-X) of method of gene recombination production, preparation Mpl with Chinese hamster ovary celI.

In addition, comprise also among the present invention that the applying gene recombinant technology prepares the method for people TPO of the present invention.People TPO preparation method for example of the present invention is characterised in that: the DNA of the people TPO that will encode is inserted into the proper site of carrier, makes this recombinant vectors and transforms or the transfection human cell line, the people TPO protein of separation, its generation of purifying.The preparation method of the people TPO that is provided in embodiment of the present invention imports TPO with people TPO gene to produce in the human cell line in internal organs source, and preferred cell (for example JHH7 cell) or the human bone marrow matrix oncocyte that derives from people's liver that import makes it to express.The human cell line that uses in these methods as mentioned above.

Conversion or the used carrier of these host cells of transfection have pSV2-neo (Southern and Berg; J.Mol.Appl.Genet., 1,327-341,1982), pCAGGS (Niwa etc., gene, 108,193-200,1991), or pCDL-SR α 296 (Takebe etc., molecular cytobiology, 8,466-472,1988) etc.

These carriers will contain replication orgin, the selected marker that is necessary, promotor is used for eukaryotic carrier and will adds necessary RNA montage position, polyadenylation signal.

Can use the replication orgin that derives from SV40, adenovirus, bovine papilloma virus as replication orgin.

Promotor or application that the promotor of using as genetic expression can be used viruses such as deriving from retrovirus, polyomavirus, adenovirus, SV40 derive from chromosomal promotor (for example, EF1-α) etc.

Can use Xin Meisu (neo) resistant gene, tetracycline (pur) resistant gene, thymidine kinase (TK) gene, Tetrahydrofolate dehydrogenase (DHFR) gene, intestinal bacteria xanthine-guanine phosphoribosyl transferase (Ecogpt) gene etc. as selective marker.

Preferably as back embodiment 1 (4), transform the JHH7 cell, be about to people TPO cDNA and be configured in people's elongation factor-1-α promotor downstream, add SV40 initial stage adenylic acid (AMP) tailing signal, DHFK gene SV40 replication origin, with such carrier, by infection protocol (Promega company) transfection JHH7 cell.Perhaps the JHH7 cell of transfection also produces the TPO based on the exogenous human TPO gene that imports except that producing endogenous people TPO expression of gene product people TPO.

In addition, the present invention also provides the pharmaceutical composition that contains people TPO of the present invention.Pharmaceutical composition of the present invention can comprise stablizer, thinner, disintegrating agent, sanitas, antacid, vehicle and the isotonic agent etc. that adapt with the preparation purpose.

The medicine of the people of comprising TPO of the present invention can be non-per os approach such as be suitable for injecting, through the formulation of number of ways such as lung, intranasal and per os, for example solution, suspension agent, tablet, pill, capsule, granule, lyophilized preparation etc.

The pharmaceutical composition that comprises people TPO of the present invention, its active component content be 0.05 μ g/kg-1mg/kg body weight normally, and according to the state of an illness, reaching route of administration especially can be once-a-day-use for several times.

The thrombocyte dose that the present invention is an effective constituent for numerous diseases patient that must platelet increasing provides with people TPO of the present invention.

Moreover, for because of using thrombocytopenia that carcinostatic agent and immunosuppressor cause, implementing the therapeutical agent that chemotherapy and radiation or the patient of bone marrow transplantation (BMT) and PBSCT, CBSCT provide thrombocytopenia.

Moreover to because of the thrombocyte obstacle, for example thrombocyte dyspoiesis and platelet life span shorten (platelet destruction is hyperfunction, or platelet consumption is hyperfunction) and cause thrombopenia to provide therapeutical agent for the multiple disease of feature.

For example, congenital fancoil's anemia, follow the hypoplastic anemia of chemotherapy and radiation method, marrow abnormal formation disease, acute myelogenous leukemia, perhaps resemble the bone marrow transplantation because of marrow forms the thrombocytopenia that do not cause entirely etc., this therapeutical agent can be owing to promoting the hematoblastic recovery of these patients.

In addition, also useful to producing the thrombocytopenia that causes unusually because of TPO.As the thrombocytopenia that causes because of thrombocyte and the megalokaryocyte lost of life, idiopathic thromboeytopenic purpura idiopathic thromboeytopenic purpura for example, acquired immune deficiency syndrome (AIDS) (AIDS), disseminated inravascular coagulation syndrome, thrombus thrombocytopenias etc., this therapeutical agent can be used for promoting these patients' platelet recovery.

Moreover bestowing TPO before the surgical operation can increase the thrombocyte of self, and this thrombocyte can be used as to transfuse blood when self performs the operation uses thrombocyte, that is to say to can be used for self platelet transfusion.

Moreover people TPO of the present invention one crosses property thrombocyte thrombocyte obstacle damaged or that damage is caused and also has therapeutic action for what cause because of other pharmaceutical chemicals or medicine or the treatment measure of living.People TPO of the present invention can be used for promoting new " do not have and hinder " hematoblastic release of these patients.Moreover, because one of main generation internal organs of clear and definite TPO are liver,, expectation causes thrombocytopenic various hepatopathy so imposing on TPO clinically, for example, and Biliary atresia disease, liver transplantation, liver cirrhosis, hepatitis etc.Moreover can be used for preserving the thrombotic ability of hematoblastic hemostasis.

Thus, use pharmaceutical composition of the present invention or therapeutical agent, not only can reduce its antigenicity better, and can improve the pharmacokinetics of its medicament better, better thrombocyte be can obtain and effect, thrombocyte treating dysfunction effect or thrombocytopenia result of treatment increased.

The present invention also provides the people TPO that has the sialylated sugar chain structure of α 2,6 connecting-types at least.Here, people TPO is that ectogenic people TPO gene is expressed in cell strain, the product of separation and purification.In addition, same with Chinese hamster ovary celI and gonad cell, can be with α 2,6-sialic acid based transferase gene imports α 2, in the cell strains such as active low HEK293 that derives from kidney of 6-sialytransferase and BHK.

The inventor in the 6-sialic acid based transferase cDNA importing Chinese hamster ovary celI, attempts expressing the TPO that adds α 2,3 connecting-types and two kinds of sialic acid forms of α 2,6 connecting-types by with rat α 2.At this moment, people TPO gene can be imported behind the Chinese hamster ovary celI again with α 2,6-sialytransferase cDNA imports, but also reverse order carries out.Or carry out simultaneously.In addition, the gene that imports to Chinese hamster ovary celI in embodiment afterwards can be rat liver beta galactose glycosides α 2,6-sialic acid based transferase (Entrez registration number M18769), for example derive from or the enzyme (Entrez registration number L29554) and the mouse beta galactose glycosides α 2 of rat brain 6-sialytransferase (Entrez registration number D16106).

Original Chinese hamster ovary celI does not have α 2,6-sialic acid based transferase activity (Paulson, J.C. etc., journal of biological chemistry, 264 volumes, 10931-10934 page or leaf, (1989)), but according to Lee, E.J. etc. (journal of biological chemistry, 264 roll up the 13848-13855 page or leaf, (1989)) method, by importing α 2 in the strong promoter downstream, 6-sialic acid based transferase cDNA, the α 2 that originally has with cell, 3-sialic acid based transferase activity is competed, according to the advantage of its amount, α 2,6 mating type sialic acids and α 2,3 connecting-type sialic acids perhaps produce the TPO that add the advantageous position on a part.When " α 2,6 mating type sialic acids add the advantageous position to " said here was meant in embodiment 3 measuring with lectin ELISA of narration, it was reflected at below the mensuration boundary with SSA-lectin ELISA reaction and MAM lectin ELISA's.The TPO that need not recognize the generation of CHO D-pearl has α 2,3 type bonded sialic acids, and does not have α 2,6 type bonded sialic acids, and in fact, it and MAM lectin ELISA have very strong reactivity, and SSA lectin ELISA is had not a particle of reaction.By importing α 2, the 6-sialic acid transferase gene can reverse this reactivity.The α 2 that reaches and do not have originally, 6-type bonded sialic acid produce the reaction of specificity bonded SSA lectin and are meant that import α 2, the 6-sialic acid transferase gene makes it multilist and reaches.Its active and α 2 as a result, the interpolation activity of the saliva of 3-sialytransferase is compared, and accounts for overwhelming majority, and the sialic acid point of addition of original α 2, the 3 type combinations of result is occupied by the sialic acid of α 2,6 type combinations.

As the Chinese hamster ovary celI that imports people TPO gene, available by the Chinese hamster ovary celI that suitable expression vector transformed that comprises coding people TPO DNA.For example can recommend the Chinese hamster ovary celI strain (CHO-DUKXB11) that transformed by the plasmid pDEF202-hTPO-P1 that keeps coding people total length TPO cDNA, this plasmid is preserved in life engineering Industrial Technology Research Institute of Japan as Represented by Director General of Agency of Industrial Science and Tec on January 31st, 1993, and preservation is FERM BP-4988.

So, the invention provides the preparation method of the people TPO that has the sialylated sugar chain structure of α 2,6 connecting-types at least, it comprises α 2, and 6-sialytransferase cDNA imports Chinese hamster ovary celI, makes the method for people TPO genetic expression in this Chinese hamster ovary celI.The invention provides the preparation method of the people TPO that has the sialylated sugar chain structure of α 2,6 mating types at least in addition, it is included in and makes people TPO genetic expression in the Chinese hamster ovary celI, and again with α 2,6-sialic acid based transferase cDNA imports the method in this Chinese hamster ovary celI.

Embodiment

By following examples the present invention is specifically described, but the present invention is not limited to these embodiment.＜embodiment 1〉(1) from human plasma (part) purifying TPO ... native plasma TPO

The human plasma of-80 ℃ of preservations of the about 460ml of dissolving in flowing water, 1350 * g is centrifugal afterwards, removes infusible precipitate, contains 1mM EDTA, 0.1%Tween 20,0.1% NaN with equivalent ₃Dulbecco ' s phosphate-buffered saline (PBS) (DPBS: day water pharmacy (strain) company catalog number 05913) dilute its supernatant 457ml.Proteolysis for the arrestin enzyme causes adds proteinase inhibitor PefablocSC (MERK company catalog number 124839, final concentration 0.1mM) in the human plasma 914ml of 2 times of dilutions.Add trans-Epoxysuccinic acid-L-leucylamino (4-guanidine radicals)-butane (SIGMA, catalog number E3132, final concentration 0.01mM).The SARTOBRAN 300 (the catalog number 5231307H5--00--B of Sartorius company) that uses 0.22 μ is with it filtration, with sample on the flow velocity of 0.5ml/min to NHS-activated Sepharose 4FF post (the post size: φ 0.5cm * 5cm that combines anti-TPO antibody, NHS-activated Sepharose FF gel: Pharmacia, catalog number 17-0906-01).This moment is for fear of the non-commentaries on classics opposite sex absorption of anti-TPO antibody column, with Sepharose 4FF post (post size: φ 0.5cm * 0.5cm, Sepharose 4FF glue: Pharmacia, catalog number 17-0149-01) and normal rabbit igg bonded NHS-activated Sepharose 4FF post (post size: φ 0.5cm * 5cm, NHS activated Sepharose 4FF glue: Pharmacia, catalog number 17-0906-01) as front pillar.Connect anti-TPO antibody column then.After the upper prop, with containing 1mM EDTA, 0.1%Tween 20,0.1%NaN ₃The DPBS damping fluid wash post, when the absorbancy (A280) of 280nm is removed front pillar when reaching 0.2.In order to remove and the different in nature adsorptive of the non-commentaries on classics of glue bonded, wash post with 10mM sodium phosphate pH 7.3 damping fluids that contain 0.5M NaCl.With after the 150mM NaCl displacement, use the 0.1M glycine-HCl pH 2.3 buffer solution elution TPO that contain 150mM NaCl, 5mM CHAPS in the post.Behind the wash-out, use 1M Na immediately ₂CO ₃In and each several part, supply with high sensitive TPO-ELISA.Select TPO wash-out part according to ELISA result, concentrate, exchange buffering liquid by ultrafiltration.Carry out ultrafiltration with ULTRAFREE-MC 10kcut (MILLIPORE, catalog number UFC3LGCOO), 4000 * g is centrifugal, after concentrating, adds DPBS for several times, concentrates repeatedly.Be suspended to 500L at last and contain 1mM EDTA 0.1%Tween 20,0.1%NaN ₃The DPBS damping fluid in, obtain partially purified human plasma TPO.

The following sds gel that carries out of a part of partially purified this TPO is filtered.The 250mM Tris-HCl, the pH6.8 that in partial purification TPO, add 5 part of 1 volume, 50% glycerine, 5%SDS, 10mM EDTA handled 5 minutes for 95 ℃.With on sample to the Superose 6HR gel-filtration column (φ 1cm * 30cm that crosses with the DPBS balance that contains 0.1%SDS, 1mM EDTA, Pharmacia, catalog number 17-0537-01), gained level part is supplied with TPO-ELISA, identify the wash-out position of TPO.The result derives from human plasma and TPO and CHO expression type TPO (1-332 amino acid) and almost goes the same position wash-out to go out, and considers the about 80KD of molecular weight.In addition, though amount approximately can see that part elongated TPO wash-out comes out near the 20KD less.

(2) from JHH7 cell (part) purifying TPO ... natural HepTPO (JHH7)

It is 225cm that the JHH7 cell is put into the sub-area of cultivation ²Culturing bottle in, with Dulbecco ' s improvement Eagle ' s substratum/HamF12 (DF) substratum (Gibco) that contains 10%FCS, at 5%CO ₂Incubator in, cultivate for 37 ℃ and converge., be replaced as the DF substratum of serum-free, cultivate after 4 days, reclaim supernatant thereafter.

With the synthetic every part of 1L of the supernatant (TPO concentration 55pg/ml) of the volume 4L that obtains in the above-mentioned cultivation, go up in the post of the WGA-agarose that crosses with 5mM potassiumphosphate pH6.8 balance (biochemical industrial society system) 5ml filling then.After cleaning with the 5mM potassiumphosphate pH6.8 of about 250ml, with 0.4M N-acetylgalactosamine, 5mM potassiumphosphate pH6.8 15ml wash-out.With UF15-10kcut (Millipore company) concentrate eluant, obtain the TPO sample (1.0ml, 159ng/ml).(3) from HuH7 cell (part) purifying TPO ... natural HepTPO (HuH7)

The HuH7 cell is placed into the sub-area 225cm of cultivation ²Culturing bottle in, with containing the DF substratum of 10%FCS, at 5%CO ₂In the incubator, 37 ℃ be cultured to and converge., change the DF substratum of serum-free, cultivate after 4 days, reclaim supernatant thereafter.After the supernatant (147pg/ml) of 1.9L is divided into every part of L.Go up to the post of WGA-agarose (biochemical industrial) the 5ml filling of crossing with 5mM potassiumphosphate pH6.9 balance.After cleaning with the 5mM potassiumphosphate pH6.8 of about 250ml, carry out wash-out with each 15ml of 0.4M N-acetylgalactosamine 5mM potassiumphosphate pH6.8.With UF15-10kcut (Millipore) concentrate eluant, obtain the TPO sample (1.5ml, 77ng/ml).(4) expression and the purifying of TPO in the JHH7 cell ... reorganization HepTPO (JHH7/7/c83)

In the plate (Pharmacia company) of diameter 6cm, with the DMEM culture medium culturing JHH7 cell that contains 10%FCS, make it propagation, use ト ラ Application ス Off エ Network system method (Promega), transform with pDEF202-h-TPO-P1 (spy opens flat 8-228781 communique).

The Chinese hamster ovary celI (CHO-DUKBII) that plasmid pDEF202-h-TPO-P1 is transformed is preserved in life chemistry Industrial Technology Research Institute of Japan as Represented by Director General of Agency of Industrial Science and Tec January 31 nineteen ninety-five, and registration number is FERM-BP-4988.

Promptly, 10 μ g pDEF202-hTPO-P1 plasmids, 1 μ g pEFneo plasmid are joined in the DMEM substratum of 500 μ l and constitute SolutionA, in 500 μ l DMEM substratum, add 5 μ l (1mg/400 μ l EtOH) and constitute Solution B, the two is mixed, in the plate of the diameter 6cm that is replaced as the big serum DMEM of 500 μ l substratum, add in 1ml, 37 ℃, 5% the incubator and placed 6 hours.Then, add DMEM substratum 3.5ml, add final concentration and reach 10% serum, again at 37 ℃, 5%CO ₂Placed 48 hours in the incubator.Being produced as follows of pEFneo plasmid carried out.Promptly, handle plasmid pRc/CMV (Invitrogen) with Restriction Enzyme EcoRI-BamHI, reclaim the small segment that contains neomycin resistance gene by agarose gel electrophoresis, with T4 polysaccharase (precious wine is made) with terminal flush end processing after, the carrier DNA that obtains is connected with the plasmid pEF18S (spy opens flat 8-228781 communique) that handles with Restriction Enzyme SmaI with this fragment with T4DNA polysaccharase (precious wine is made).In 2 kinds of plasmids of gained like this, the plasmid of selecting the same direction of neomycin resistance gene and elongation factor promotor to insert is pEFneo.

With 0.05% trypsinase, 53mM EDTA solution it is peeled off dispersion then, be scattered in 10 24 orifice plates after, screen with the DMEM+10%FCS substratum that contains 4mg/mg G418.After wherein forming colony, with the colony of FDCPhMp1635/MST identification method (Morita H. etc., FEBS Lett.395 volume, 228-334 page or leaf, (1993)) screening high expression level.With limiting dilution assay the colony that screens is carried out cloning, as monospecific polyclonal.

The JHH7 cell strain (JHH7/7/c83) that comprises the pDEF202-hTPO-P1 conversion of people TPO cDNA is preserved in Japan's trading industry on August 11st, 1997 according to budapest treaty and economizes Industrial Technology Institute life engineering Industrial Technology Research Institute, and preserving number is FERM BP-6050.

It is 225cm that the JHH7 cell strain (JHH7/7/c83) of should recombinating is put into culture area ²Culturing bottle in, with containing the DF substratum of 10%FCS, at 5%CO ₂37 ℃ are cultured to and converge in the incubator., it displacement to the DF substratum of serum-free, cultivated 4 day after, reclaim supernatant thereafter.

With the supernatant simmer down to 899ml (TPO concentration be 5.7 μ g/mls) of Pellicon-2cassette 10kcut (Millipore company, lot number P2B010A05) with 8.97L.For the albumen aqua of arrestin enzyme, add the bright protein peptide (Leupeptin) (final concentration 0.01mm), trans-epoxy succinic acyl-L-leucylamino (4-guanidine radicals)-butane (final concentration 0.01mm) that presses down of proteinase inhibitor.

Be divided into every part of 225ml, go up on the post of 4 WGA0-agavose (biochemical industrial) 5ml fillings of crossing with 5mM potassiumphosphate pH6.8 balance in advance.After the 5mM potassiumphosphate washing with about 250ml, carry out wash-out (790 concentration are 73 μ g/ml) respectively with 0.4M N-ethanoyl GalN, each 15ml of 5mM potassiumphosphate pH6.8.

With UF15-10kcut (Millipore) concentrate eluant, be replaced as 5mM potassiumphosphate pH6.8 (5.0ml, TPO concentration 1084 μ g/ml).

It is injected Hydroxyl Apatite typel (Biorad, diameter 1cm, the high 10cm of bedding) post, after adding with the flow velocity of 1ml/min, continuously 5nm potassiumphosphate pH6.8 is injected in the post, collect the not part 50ml (TPO concentration 54 μ g/ml) of absorption.To wherein adding 1M Tris alkaline solution, adjust pH is 8.5.

Then, with Q-Sepharose HP (Pharmacia Biotech, lot number diameter 5mm, the high 10cm of bedding) post launches this part, promptly launch solvent orange 2 A 20mM Tris-HCl pH7.5, launch solvent B with the 20mM Tris-HClpH 7.5 that contains 1M NaCl, on the pillar of the sample additive being crossed with 20mM Tris-HCl pH of buffer 7.5 balances in advance with the flow velocity of 0.3ml/min.After adding end of a period, flow velocity transfers to 0.5ml/min from 0.3ml/min, launches with the straight line concentration gradient of 0%B-22%B, and per 1.0 μ l (2min) collect in the pipe of polypropylene system.

Measure at different levels parts with the ELISA method, identify TPO level part.Found that the TPO that has that test tube is numbered 34-55 (NaCl concentration is 0.116-0.2M) distributes, and therefore reclaims this part, as TPO level part FA (22ml).Concentrate this part with UF15-10kcut (Millipore), when liquid measure reaches 4.1ml, measure the concentration (1143 μ g/ml) of TPO with the ELISA method.Further concentrate again, make it to reach 200 μ l.

Final step is to separate with Superdex 200HR (Pharmacia Biotech, diameter 1cm, height of bed 30cm).That is, post is replaced as phosphate-buffered saline (PBS) after, FA concentrating sample 200 μ l are injected with the flow velocity of 0.3ml/min.Add pillar with PBS continuously after the injection, add beginning from sample and begin per 100 μ l (3min) after 15 minutes as one-level part.Clonal antibody, ELISA method by silver dyeing, anti-people TPO are measured at different levels parts, and found that the test tube numbering 13,14,15 (is 7.2-9.0 μ l from the total amount of adding beginning) has TPO to distribute.Merge this 3 level parts, analyze its composition with the AccQTag method, the result is people 1.733mg/ml (1.8ml) fraction.

Compositional analysis value when at this moment, being 1.1mg/ml with the TPO amount of measuring with the ELISA method much at one.That is to say, even if applied here ELISA also shows the equal reactivity with standard TPO to reorganization HepTPO.

Table 1 reorganization HepTPO (JHH7/7/c83)

CBB method ELISA

Sample	Fraction	Volume (ml)	Protein concentration (mg/ml)	Total amount (mg)	Flow velocity	TPO concentration (μ g/ml)	Total amount (μ g)	Reclaim (%)
									?Cond. ?Med		????8971	????0.09408	????843.99168	????100.0	????0.55	??4934.05	????-
?Conc. ?Med		????899	????0.9298	????835.8902	????99.0	????5.7	??5124.3	????100.0
									?WGA	?FT	????903	????0.8693	????784.9779	????93.0	????0.45	??406.35	????7.9
	?Ads	????60	????0.3448	????20.688	????2.5	????72.95	??4377	????85.4
										?Conc	????5		????0	????0.0	????1084.15	??5420.75	????105.8
?HA	?FT	????50		????0	????0.0	????54.4	??2720	????53.1
									?Q	?pool	????4.1		????0	????0.0	????1143	??4686.3	????91.5
?Superdex	?pool	????1.8		????0	????0.0	????1127.85	??2030.13	????39.6

AccQ-TAG method (mg/ml)

1.11733＜embodiment 2〉mensuration of molecular weight

In order to measure the molecular weight of the various TPO that obtain among the embodiment 1, carry out the SDS-PAGEWestern trace.According to ordinary method, use under micropore gel (the first pharmaceutical chemicals company, the polyacrylamide gel of the 4.0%-20% concentration gradient) room temperature and carry out sds gel electrophoresis, with first 10mA, the electric current of 20mA carried out about 2 hours then.Use pre-staining wide region mark as molecular weight marker.

Use half-dried transfer device (マ リ ソ Le company, ウ エ Star ト Off オ transfer device KS-8640 type), electrophoresis stop the back directly the electric current of usefulness 150mA through 7 hours electrotransfers to PVPF film (Millipore).Anode 0.3M Tris, 20% methyl alcohol, pH10.4 changes film liquid 25mM Tris, 20% methyl alcohol, pH10.4, the amino n-caproic acid of catholyte 25mM Tris, 40mM, 20% methyl alcohol.

The film that shifts washed 5 minutes in TTBS solution after, in confining liquid (Seichin society), sealed 1 hour.After in TTBS, washing 5 minutes, in 90%TTBS, 10% confining liquid, 0.03%BSA solution, add anti-people TPO goat-anti body, vibrated 1 hour.Then, by in TTBS this film being washed 2 times, each 3 minutes, the anti-sheep peroxidase binding antibody of adding in TTBS solution vibrated 1 hour then., this film in TTBS solution washed 4 time, each 10 minutes, in ELC Color Appearance System (Amersham), make film (Amersham) sensitization thereafter.

The molecular weight of being seen in the electrophoresis, almost all there be (Fig. 1 and Fig. 2) in recombinant C hoTPO (CHO), natural HepTPO (JHH7, HuH7), reorganization HepTPO (JHH7/7/c83) near 80KDa.This is that (35KDa has very big-difference, and can release this at an easy rate is because it has added sugar molecular weight to be increased with the molecular weight of calculating from aminoacid sequence.＜embodiment 3〉use lectin-ELISA and carry out the sialic acid analysis

With the present reported method (Rafferty of institute, B. etc., J.Endoeri, 145 volumes, 527-533 page or leaf) similar, lectin ELISA is by using the specific antibody relative with target protein matter as solid phase, with absorption target protein matter, make it to react with biotinylated lectin, then the avidin with mark reacts, colour developing is undertaken by measuring absorbancy.

That is, under the concentration of 10 μ g/ml, anti-people's mouse monoclonal antibody, TN-1 (Tahara, J. etc., Britain's hematology magazine, 57 volumes, 783-788 page or leaf (1995)) are added to respectively in each hole of 96 orifice plates with every hole 100 μ l, stir the back and placed about 10 hours.Each hole with the TTBS solution of 350 μ l with it washing 4 times, in each hole, add then 200 μ l in advance with identical lectin bonded agarose (SSA-Agarose or the MAM-Agarose that are used to detect, be biochemical industrial) put upside down the 1%BSA/TTBS solution that mixes 4 hours, removed the lectin reactant, stirring at room 1 hour.Abandon it then, after dry 30 minutes, in aforesaid 1%BSA/TTBS solution, add the sample of various concentration, every concentration 3 holes, each hole 100 μ l stirred 4 hours.By washing plate in the TTBS solution of going into each hole 350 μ l, after washing 5 times, (biochemical industrial) joins in the 0.5%BSA/TTBS solution with lectin-vitamin H, final concentration is 0.2 μ g/ml during the SSA-vitamin H, final concentration is 1.0 μ g/ml during the MAM-vitamin H, each hole adds 100 μ l, stirs 2 hours.In the TTBS solution of each hole 350 μ l, wash plate once more, wash 5 times after, the concentration with each 1 μ l/100ml before 30 minutes has been added avidin-alkaline phosphatase/avidin-biotin solution (DAKO).The 0.25%BSA/TTBS solution that has stirred is added to each hole with the every hole of 100 μ l branch, stirs 1 hour.At last, in the TTBS solution of every hole 350 μ l, wash plate again, wash 6 times, add the substrate solution 100 μ l of alkaline phosphatase colouring reagents box (DAKO), act on 10 minutes, then add 100 μ l enhanced sensitivities colour developing liquid, stirred 5 minutes, and, measured control value in the 630nm place with the absorbancy of dull and stereotyped detector (biochemical industrial) mensuration 492nm, the temperature of drawing absorbancy with this value and concentration relies on curve, that is to say and depicts the various reactivities to lectin of TPO.

Native plasma TPO, natural HepTPO (JHH7) and natural HepTPO (HuH7) all have similar trend to the reactivity of SSA lectin ELISA as can be seen from Figure 3.Especially native plasma TPO, natural HepTPO (JHH7) and natural HepTPO (HuH7) show hyperergy.On the other hand, recombinant C hoTPO is detecting below the boundary.Native plasma TPO, natural HepTPO (JHH7) and natural HepTPO (HuH7) have similar trend to the reactivity of MAM-lectin ELISA, and the reactivity of reorganization HepTPO and HepTPO (JHH7/7/c83) is low slightly.＜embodiment 4〉activity in vivo

It is that various detections are administered to mouse that activity in vivo calculates principle, calculates by calculating platelet count.

That is, the various detection things that are suspended in the various concentration among the PBS were applied to the BALB/c mouse in male 8 ages in week in continuous 4 days.Continuous administration finishes the 3rd day blood-sample withdrawal in back and measures platelet count.This moment 100mn is defined as that platelet count is 3 * 10 in the blood ⁶The concentration of thrombocyte 1 μ l.

Find that with above-mentioned measuring method comparison TPO reorganization HepTPO is 4.5 * 10 when various ⁴U/mg is than 9.0 * 10 of reorganization ChoTPO ⁴The value of U/mg is lower.＜embodiment 5〉external activity (FDCP-hMp1635 mensuration)

Reclaim FDCP-hMp1635 cell (Morita, H. etc., the FEBS Lett.395 volume that there be forced expression total length people Mpl in the FDC/P2 cell of cultivating that goes down to posterity down in TPO, the 228-334 page or leaf, (1995)), behind the thorough washing, be re-suspended in the IMDM nutrient solution that contains 10%FCS.With the FDCP-hMp1635 cell with every hole 2.5 * 10 ³Individual cell count is added to tissue culture with in the flat culture plate in 96 holes, adds standard substance and sample to be checked again, makes whole liquid measure reach 200 μ l/ holes.With culture hole in 5%CO ₂, 37 ℃ cultivated 3 days.Cultivate 4 hours each holes of forward direction at the 3rd day and add 25 μ l MTS/PMS solution (Promega), cultivate 492nm is measured in the back of ending with dull and stereotyped reading (biochemical industrial) absorbancy.

Analyze compositional analysis value, (Tahara, T etc., the Britain's hematology magazine of the recombinant type TPO of the TPO that derives from various cells once more, 57 volumes, 783-788 page or leaf, (1995)) derive from the ELISA value of the TPO of blood plasma or difficult recombinant type liver cell, find that they have same isoreactivity (Fig. 4).＜embodiment 6〉α 2,6-sialic acid based transferase expression vector (pEFneo-α 2, preparation 6ST)

After fully cutting off pEFneo plasmid among the embodiment 1 (4) with XbaI, the short period of time obtains the partially digested fragment of XbaI-XhoI with the XhoI effect continuously.At this moment, because there are 3 in the XhoI position, can there be 3 kinds so contain the fragment of SV40 replication orgin.Wherein the purpose fragment length the 2nd, at first 3 kinds mixture and purpose fragment are linked, then with near the primer 5 of the position of EF-1 α promotor 3 ' end of coding pEFneo '-CCTCAGACAGTGGTTCAAAG-3 ' (sequence number 2) checks order, to get rid of other possibility of 2 kinds, in fact also like this.Here the purpose fragment of being said is to contain the beta galactose glycosides α 2 that derives from rat liver, 6-sialytransferase cDNA (GenbankM18769/Weinstein, J. etc., journal of biological chemistry, 262,17733-17743) 5 of total length ' end has XhoI position, 3 ' end that the dna fragmentation at XbaI position is arranged.

This carrier contains the replication initiation zone of SV40, people's elongation factor-1-2 promotor, the initial polyadenylation of SV40 position, and SV40 promoter region, neomycin resistance gene, the initial polyadenylation of SV40 position, β-lactamase gene (Ampr), beta galactose glycosides-α 2,6-saliva transferring enzyme cDNA are connected to people's elongation factor-1-α promotor downstream.＜embodiment 7〉α 2,6-sialytransferase expression vector is to the importing of Chinese hamster ovary celI

With the CHO29/14 strain of expressing human total length TPO (200nM methotrexate resistance), with 3 * 10 ⁵Cell, place the plate (Falcon) of 6cm bore, with the necessary substratum (α-MEM (-) of the minimum that contains 10% foetal calf serum, interpolation gland thymidine, xanthoglobulin are cultivated, make it propagation, use ト ラ Application ス Off エ Network system method (Promega) it is transformed, import α 2,6-sialytransferase expression vector.

Promptly, in pEFneo-α 2, the 6 ST plasmids 15 μ g of embodiment 6 preparations, add 500 μ l α-MEM (-) and be prepared as mixture, from the 400 μ l ethanol that dissolved 1mg ト ラ Application ス Off エ Network system, get 5ul then, 500 μ l α-MEM (-) are joined wherein, be prepared as mixture.With two kinds of top mixture remix, room temperature was placed 10 minutes.Therebetween, the above-mentioned cell exchange that will cultivate on 6cm diameter plate is in the α-MEM (-) that does not contain foetal calf serum.Drip in the plate this dna solution back at CO ₂Cultivated about 6 hours in the incubator.In plate, add the α-MEM (-) of 2.5ml and the foetal calf serum of 500 μ l, continue to cultivate after 1 day, select substratum (α-MEM (-) does not add thymidine, and xanthoglobulin adds Geneticin (Giboco)) to screen with containing 10% dialysis foetal calf serum.The enforcement of screening is, with behind the trypsin treatment cell, the plate of each 6cm diameter cut apart in 10 24 orifice plates, and after 2-3 days, the selection substratum is changed on the limit, and the limit continues to cultivate.At this moment, initial 2 days with containing the selection substratum that final concentration is the 0.5mg/ml Geneticin, follow 3 days with containing the selection substratum that final concentration is the Geneticin of 1mg/ml, thereafter, when whenever changing nutrient solution, change the selection nutrient solution that Geneticin concentration reduces by half into, the concentration of Geneticin obtains 0.2mg/ml the most at last.To maintain 200mM constant for the concentration of methotrexate therebetween.For cell proliferation the hole, detect the amount of people TPO in its culture supernatant with TPO-ELISA, measure the sialic relative increasing amount of being added with α 2,6 type combinations by SSA-lectin ELISA.To seeing the active and α 2 of people TPO in the culture supernatant, each hole that 6 mating type sialic acids increase, separately be stripped to cell in the hole with the selection substratum that contains 200mM methotrexate, 0.2mg/ml Geneticin with 1: 15 again, by continuing cultivation, make Geneticin resistant cell propagation, cloning, the result finally obtains the clone CHO+2 to 200mM methotrexate 0.2mg/ml Geneticin resistance, 6ST/19/c4 and CHO+2,6ST/21/c3.＜embodiment 8〉affirmation expressed

In order to measure the molecular weight of the various TPO of gained among the embodiment 7, carry out SDS-PAGE Western trace with the method among the embodiment 2.Method (Britain's hematology magazine with reference Tahara.T etc., 57 volumes, the 783-788 page or leaf, (1995)) measure the expression amount that obtains and be the basis, the DCRP CHO+2 of institute, molecular weight that the TPO that 6ST/19/c4 and CHO+2,6ST/21/c3 produce sees in electrophoresis and recombinant C hoTPO (CHO29c14) almost coexist (with reference to Fig. 5) near 80 KDa.This and the molecular weight amount 35KDa that raises according to aminoacid sequence difference to some extent can infer at an easy rate that this is that molecular weight increases because add sugar chain.＜embodiment 9〉usefulness lectin-elisa assay sialic acid

With the method among the embodiment 3, the TPO that obtains among the embodiment 8 is carried out sialic acid analysis (Fig. 6).

The result shows, CHO+2 in the 2 strain cells, and 6ST19/c4 adds α 2 to be created in the advantageous position, the sialic acid TPO of 6-combination is the cell of feature, CHO+2, and 6ST/21/c3 produces α 2, the sialic acid of 3-and α 2,6 combinations is added on simultaneously with the TPO on a part.＜embodiment 10〉affirmation of external activity

External activity with the TPO that obtains in the affirmation of the FDCP-hMp1635 assay method among the embodiment 5 enforcement.

2.5 * 10 ³Individual FDCP hMp1635 cell adds 20 μ l MTS solution with TPO 37 ℃ of cultivations in 96 orifice plates of various concentration after 3 days in each hole.After cultivating 4 hours again, measure the activity that absorbancy is come test sample at the 492mm place.The result as shown in Figure 7.

The result shows, the TPO of CHO D-cell expressing and imported α 2, and the TPO of the expressing cho cell of 6-sialic acid transferase gene is almost equal to the proliferation activity of FDCP hMp1635.In a word, can say so: although sialic mode of connection is varied to α 2,6 types by α 2,3 types of seeing in the original Chinese hamster ovary celI, no matter ratio how, the proliferation activity of cell is identical.

Sequence table＜110〉Kirin Brewery Company.Limited＜120〉human thrombopoietin produced with cell line, its preparation method, and comprise this hematoblastic pharmaceutical composition.＜130〉PH-570-PCT＜150〉JP97/236109＜151〉1997-09-01＜160〉2＜210〉1＜211〉332＜212〉protein＜213〉people＜400〉1 Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu, 15 10 15 Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val

         20                  25                  30 His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu

     35                  40                  45 Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu

 50                  55                  60 Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln  65                  70                  75                  80 Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln

             85                  90                  95 Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu

        100                 105                 110 Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe

    115                 120                 125 Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu

130                 135                 140 Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala 145                 150                 155                 160 Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn

            165                 170                 175 Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr

        180                 185                 190 Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile

    195                 200                 205 Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln lle Pro Gly

210?????????????????215?????????????????220Tyr?Leu?Asn?Arg?Ile?His?Glu?Leu?Leu?Asn?Gly?Thr?Arg?Gly?Leu?Phe225?????????????????230?????????????????235?????????????????240Pro?Gly?Pro?Ser?Arg?Arg?Thr?Leu?Gly?Ala?Pro?Asp?Ile?Ser?Ser?Gly

245?????????????????250?????????????????255Thr?Ser?Asp?Thr?Gly?Ser?Leu?Pro?Pro?Asn?Leu?Gln?Pro?Gly?Tyr?Ser

260?????????????????265?????????????????270Pro?Ser?Pro?Thr?His?Pro?Pro?Thr?Gly?Gln?Tyr?Thr?Leu?Phe?Pro?Leu

275?????????????????280?????????????????285Pro?Pro?Thr?Leu?Pro?Thr?Pro?Val?Val?Gln?Leu?His?Pro?Leu?Leu?Pro

290?????????????????295?????????????????300Asp?Pro?Ser?Ala?Pro?Thr?Pro?Thr?Pro?Thr?Ser?Pro?Leu?Leu?Asn?Thr305?????????????????310?????????????????315?????????????????320Ser?Tyr?Thr?His?Ser?Gln?Asn?Leu?Ser?Gln?Glu?Gly

325 330＜210〉2＜211〉20＜212〉DNA＜213〉artificial sequence＜400〉2cctcagacag tggttcaaag 20

Claims (37)

1. the human thrombopoietin (TPO) that produces of human cell line.

2. the people TPO of claim 1 is characterized in that, people TPO is the expression product of ectogenic people TPO gene in human cell line, and product is through separation, purifying.

3. the people TPO of claim 2 is characterized in that, human cell line is to derive from the human cell line that TPO produces internal organs.

4. the people TPO of claim 3 is characterized in that, derives from the cell that TPO produces the human cell line behaviour liver source of internal organs.

5. the people TPO of claim 4 is characterized in that, the cell that derives from people's liver is JHH7 (FERM BP-1049).

6. the people TPO of claim 4 is characterized in that, the cell that derives from people's liver is HuH7 (FERM BP-6048).

7. the people TPO of claim 3 is characterized in that, the human cell line that derives from TPO generation internal organs is the human bone marrow matrix oncocyte.

8. the people TPO of claim 1 is characterized in that, people TPO is the expression product of endogenic people TPO gene in human cell line, and product is through separation, purifying.

9. the people TPO of claim 8 is characterized in that, human cell line is to derive from the human cell line that TPO produces internal organs.

10. the people TPO of claim 9 is characterized in that, the human cell line that derives from TPO generation internal organs is the cell that derives from people's liver.

11. the people TPO of claim 10 is characterized in that, the cell that derives from people's liver is JHH7 (FERM BP-6049).

12. the people TPO of claim 10 is characterized in that, the cell that derives from people's liver is HuH7 (FERM BP-6048).

13. the people TPO of claim 9 is characterized in that, the human cell line that derives from TPO generation internal organs is the human bone marrow matrix oncocyte.

14. the people TPO in the arbitrary claim of claim 1-13 is characterised in that altogether, it has the sugar chain structure that affinity is arranged with SSA lectin post.

15. the people TPO of claim 14 is characterized in that, the aminoacid sequence of people TPO is the 1-332 of sequence number 1.

16. the people TPO in the arbitrary claim of claim 1-13 is characterized in that, it has the sialylated sugar chain structure of α 2,6 connecting-types at least.

17. the people TPO in the claim 16 is characterized in that, the aminoacid sequence of people TPO is the 1-332 in the sequence number 1.

18. a pharmaceutical composition, it makes effective constituent with the people TPO in the arbitrary claim of claim 1-17.

19. a thrombocyte dose, it with the people TPO in the arbitrary claim of claim 1-17 as effective constituent.

20. thrombocyte treating dysfunction agent, it makes effective constituent with the people TPO in the arbitrary claim of claim 1-17.

21. a thrombopenia Remedies, it makes effective constituent with the people TPO in the arbitrary claim of claim 1-17.

22. the thrombocyte dose in the claim 19, its antigenicity is lowered.

23. the thrombocyte treating dysfunction agent of claim 20, its antigenicity is lowered.

24. the thrombopenia Remedies of claim 21, its antigenicity is lowered.

25. the thrombocyte dose of claim 19, it has improved intravital pharmacokinetics.

26. the thrombocyte treating dysfunction agent of claim 20, it has improved intravital pharmacokinetics.

27. claim 21 the thrombopenia Remedies, it has improved intravital pharmacokinetics.

28. the preparation method of a people TPO, it is included in and expresses exogenous human TPO gene in the human cell line that derives from TPO generation internal organs.

29. the preparation method of a people TPO, it is included in and expresses exogenous human TPO gene in the cell that derives from people's liver.

30. the preparation method of a people TPO, it is included in and expresses exogenous human TPO gene in the human bone marrow matrix oncocyte.

31. the preparation method of a people TPO, it is included in and expresses exogenous human TPO gene among the JHHT (FERM BP-6049).

32. the people TPO that obtains by the method in the arbitrary claim of claim 28-31.

33. contain the people TPO of α 2,6 mating type saliva acidizing sugar structures at least.

34. the people TPO of claim 33 is characterized in that, it is the expression product of ectogenic people TPO gene in cell strain, separated, the purifying of product.

35. the people TPO of claim 34 is characterized in that, above-mentioned cell strain is to have imported α 2, the Chinese hamster ovary celI of 6-sialic acid based transferase gene.

36. have the preparation method of the people TPO of the sialylated sugar chain structure of α 2,6 connecting-types at least, it comprises that with α 2 6-sialic acid based transferase group imports Chinese hamster ovary celI, expressing human TPO gene in this cell.

37. have the preparation method of the people TPO of the sialylated sugar chain structure of α 2,6 connecting-types at least, it is included in expressing human TPO gene in the Chinese hamster ovary celI, then with α 2,6-sialic acid based transferase cDNA imports in this Chinese hamster ovary celI.

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